Characterization of Interpolyelectrolyte Complexes between Double

Shoichiro Asayama, Takashi Sekine, Hiroyoshi Kawakami, and Shoji Nagaoka ..... Won Jong Kim , Tsutomu Ishihara , Toshihiro Akaike , Atsushi Maruyama...
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Bioconjugate Chem. 1998, 9, 292−299

Characterization of Interpolyelectrolyte Complexes between Double-Stranded DNA and Polylysine Comb-Type Copolymers Having Hydrophilic Side Chains Atsushi Maruyama,* Hiromitsu Watanabe, Anwarul Ferdous, Maiko Katoh, Tsutomu Ishihara, and Toshihiro Akaike Department of Biomolecular Engineering, Faculty of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta, Midori, Yokohama 226-8501, Japan. Received August 11, 1997; Revised Manuscript Received January 29, 1998

The polyionic interaction between DNA and polycations grafted with hydrophilic dextran side chains was evaluated. The comb-type copolymers, poly(L-lysine)-graft-dextran, were successfully prepared by employing a reductive amination reaction between -amino groups of poly(L-lysine) (PLL) and the reductive ends of dextran (Dex). A coupling efficacy on the order of 70% was obtained regardless of intrinsic philicities of the solvents used, either aqueous buffer or DMSO. The resulting graft copolymers, which varied in the degree of grafting and the length of hydrophilic side chains, formed a soluble complex with DNA. They also affected the melting behavior of double-stranded DNA (dsDNA) in different ways. Copolymers having a high degree of grafting thermally stabilized dsDNA without affecting its reversible transition between single-stranded and double-stranded forms. However, copolymers with a low degree of grafting or with a high degree of grafting of short dextran chains impeded the reversibility of this transistion. Furthermore, highly grafted copolymers also accelerated the hybridization of DNA strands in a low-ionic strength medium. It is of particular note that these copolymers scarcely altered circular dichroismic signals of dsDNA even when the copolymers were added in excess. This suggested that the copolymer interacted with dsDNA without affecting its native structure or physicochemical properties. Finally, the copolymer even formed a stable complex with a short oligonucleotide (20 bases). We, therefore, concluded that, by regulating the degree of grafting and the molecular weight of grafted side chains, it would be possible to design novel different graft copolymers capable of acting as carriers of functional genes to target cells or tissue.

INTRODUCTION

Polycations interact with polyanions strongly to form polyion complexes (PICs) or interpolyelectrolyte complexes (IPECs). Although both polyanions and polycations are readily soluble in water, when combined in solution they form complexes, such as coacervates or precipitates, which separate out of aqueous media (14). IPECs have been investigated as biocompatible (58), membrane- (5) and enzyme-immobilization (9), and microencapsulation (10-12) materials. More recently, complexes between DNA and polycation derivatives have been used as artificial carriers of poly(oligo)nucleotides, including genes and antisense oligonucleotides (13). However, applications of IPECs are limited due to their irreversible and insoluble behavior. As a result, regulation of both properties and structures of IPECs is needed to expand the functional applications of these polymer complexes. While complex formation in mixtures of linear polyanions and polycations has frequently been observed, little is known about IPEC formation between polyion segments in mixed block (14-16) or graft copolymers. In specific, complex formation is poorly understood between graft copolymers of one polyion and polymers of the opposite charge (17, 18). We previously reported that the graft copolymer consisting of a poly(L-lysine) (PLL) * Corresponding author. Phone: +81-45-924-5809. Fax: +8145-924-5815. E-mail: [email protected].

backbone and dextran (Dex) graft chains forms a soluble complex with DNA and stabilizes DNA triple helices of poly(dA)‚2poly(dT) (17). Without the copoloymer, these triple helices were unstable under physiological conditions (19). Despite the graft copolymers’ considerable elevation of the triple helix melting temperature (Tm), they did not disturb reversible transition between the triplex and its constituent single-stranded DNAs (ssDNAs). The main aim of this study was to understand and to control the interaction between polyanionic DNA and various graft copolymers consisting of a PLL backbone and hydrophilic graft side chains of Dex. PLL-graftDex (PLL-g-Dex) copolymers with various degrees of grafting and lengths of Dex chains were synthesized, and their interaction with DNA was evaluated by turbidity assays, UV-melting temperature assays (UV-Tm), circular dichroism (CD) measurements, and gel electrophoresis assays. We found that the graft copolymers formed basically two categories of soluble complexes. The first category of graft copolymer-DNA complexes allowed the reversible transition between duplex and its ssDNAs. Such copolymers were also capable of thermal stabilization of dsDNA. The second category included mostly irreversible complexes. Our results suggest that, by changing the molecular weight, degree of grafting, and the properties of the graft chains, we were able to control the associating properties of these polyions.

S1043-1802(97)00151-1 CCC: $15.00 © 1998 American Chemical Society Published on Web 02/24/1998

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Table 1. Preparation of PLL-graft-Dex Copolymersa in feed PLL run 1 2 3 4 5 6 7 8 9 10 11 12

b/104

Mn

3.6 3.6 3.6 3.6 3.6 3.6 3.6 3.6 3.6 3.6 1.3 1.3

b

mg

Mn

92 92 92 92 92 92 92 62 62 62 31 31

2600 2600 2600 2600 5900 5900 5900 5900 5900 5900 5900 5900

copolymer

dextran mg wt % 94 187 375 937 212 425 850 150 300 600 300 450

50.6 67.1 80.3 91.1 69.8 82.2 90.3 70.8 82.8 90.6 90.9 93.6

NaBH3CN (mg)

yield (%)

22.7 45.0 91.0 227 22.7 45.0 91.0 7.5 15.0 30.0 15.0 22.5

26 78 66 71 62 nd 71 60 77 79 69 74

molecular weight Mnb/104 Mw/Mnb 8.2 9.5 11.2 22.7 8.3 13.5 30.5 nd nd nd 15.7 19.6

1.5 1.4 1.7 1.4 1.6 1.4 1.2 nd nd nd 1.2 1.2

dextran contentd wt % mol %d 43.7 60.3 74.3 87.3 66.0 79.3 88.3 70.9 83.0 90.7 90.4 92.6

3.8 7.5 14.2 34.0 3.6 7.1 14.0 4.7 9.6 19.7 17.0 21.4

coupling efficacye (%) 76 75 71 68 72 71 70 88 90 93 82 68

a The reaction was carried out in the borate buffer (runs 1-7) or in DMSO (runs 8-12). b M ) the number-average molecular weight; n Mw ) the weight-average molecular weight. Mn and Mw were determined as described in Materials and Methods. c Determined by 1H NMR. d Mol % ) 100(fraction of lysine units coupled with Dex). e Coupling efficacy ) [Dex]copolymer/[Dex]in feed.

Figure 1. Structural formula of the polylysine-graft-dextran copolymer. MATERIALS AND METHODS

Materials. Poly(L-lysine) (PLL) was purchased from Sigma-Aldrich Japan (Tokyo, Japan) or Peptide Institute, Inc. (Tokyo, Japan). Dextran T-10 (Mn ) 5900), poly(dA), and poly(dT) were obtained from Pharmacia Biotech (Uppsala, Sweden). Oligonucleotides were a HPLCpurified grade obtained from Grainer Japan Co. (Tokyo, Japan). Dextran 4 (fraction with a MW of 4000-6000) was purchased from Funakoshi Chemical Co. (Tokyo, Japan). Calf thymus DNA (t-DNA) was obtained from Sigma-Aldrich Japan. Other solvents and chemicals were reagent grade, were obtained from Wako Pure Chemical Industries, Ltd. (Osaka, Japan), and were used without further purification. Synthesis of the Poly(L-lysine)-graft-Dextran Copolymer. Comb-type copolymers, poly[(L-lysine)-graft(R-1,6-D-glucopyranoside)] (PLL-g-Dex), were synthesized by a reductive amination reaction of PLL‚HBr with dextran (Dex) (17, 20). Either borate buffer or DMSO was used as a solvent for the reaction. The conditions for the synthesis are described in Table 1. The resulting copolymers were isolated by ultrafiltration to remove unreacted Dex. The ultrafiltration was carried out using a Millipore UFV2BGC10 centrifugal filter device (MW cutoff of 10 000, Millipore Corp., Bedford, MA) or a Q0100 ultrafilter (MW cutoff of 10 000, Advantec Toyo Filter Ltd., Tokyo, Japan). Finally, the copolymers were lyophilized. 1H NMR of the copolymers in D O was recorded on a 2 Varian VNMR 4.3 spectrometer and used to determine the composition of the resulting copolymers. The structural formulas of the comb-type copolymer are shown in Figure 1. Hereafter, we designate the copolymers as PLL-Dex(X-Y), where X and Y represent the molecular weight (number average) and the content (mole percentage) of Dex chains in the copolymers, respectively.

Gel Permeation Chromatography (GPC) and Molecular Weight Determination of Polymers. The resulting copolymers were analyzed using a gel permeation chromatography system (GPC, Jasco model 800, Tokyo, Japan) on Waters Ultrahydrogel 250 and 500 columns connected with a multiangle light scattering detector (Dawn-DSP, Wyatt Technology, Santa Barbara, CA) and a differential refractive index (RI) detector (Jasco, model 830-RI). An aqueous solution of 0.5 M acetic acid and 0.2 M Na2SO4 was used as a mobile phase at 1.0 mL/min. An aliquot (200 µL) of graft copolymer solution (1 mg/mL) was injected into the column. The molecular weight and its distribution were determined according to the instruction manual of Dawn-DSP on the basis of Zimm’s equation (eq 1).

Rθ ) MP(θ) - 2A2cM2P2(θ) K*c

(1)

where c is the concentration of solute molecules in the solvent r, M is the weight-average molecular weight, A2 is the second virial coefficient, P(θ) is a theoretically derived factor and is a function of the molecule’s zaverage size, shape, and structure, R(θ) is the Rayleigh ratio at the individual scattering angle, and K* is an optical constant [)4π2n02(dn/dc)2λ0-4NA-1, where n0 is the refractive index of the solvent at the incident radiation wavelength (nanometers), NA is Avogadro’s number, and dn/dc is the differential refractive index increment of the solvent/solute solution with respect to a change in solute concentration]. By extrapolating the value of θ to 0, we derived eq 2 from eq 1.

Rθf0 ) M - 2A2cM2 K*c

(2)

By neglecting the second term, we detemined M with a Rθ/K*c versus sin2(θ/2) plot (Debye plot) for θ approaching 0. M was calculated at each second of the elution period using c monitored by the RI detector. The estimated values of M at each 1 s slice in the GPC were then used to determine weight- and number-average molecular weights of polymers. Turbidity Measurements of the DNA/Copolymer Mixture. Calf thymus DNA was dissolved in Milli Q water at 1.4 mg/mL and sonicated twice for 8 min on ice with a 80 W output at a 20% interval using a probe-type generator (Tomy UD-201, Tokyo, Japan). The approxi-

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mate molecular weight of the sonicated DNA fragments was 1 kbp as determined by agarose gel electrophoresis (data not shown). The DNA solution was diluted to 30 µg/mL with 10 mM sodium phosphate buffer solution (PBS, pH 7.2) containing 150 mM NaCl (buffer I). To the DNA solution was added a stock solution (1 mg/mL) of the graft copolymer in buffer I at various polymer/DNA charge ratios (P/D ) [amino group]polymer/[phosphate group]DNA). The final DNA concentration was adjusted to 27.4 µg/mL. After the solution was vigorously mixed with a vortex mixer, it was allowed to stand at 4 °C. Turbidity measurements were carried out after 1, 4, and 14 days at 340 nm with Beckman DU 640 spectrophotometer with a 1 cm path length. CD and UV-Tm Measurements. Stock solutions were prepared by dissolving nucleotides in 10 mM PBS (pH 7.2) containing 150 mM NaCl and 0.1 mM EDTA (buffer II). Concentrations of the stock solutions were calculated using molar extinction coefficients of 8900 at 257 nm for poly(dA) and 9000 at 265 nm for poly(dT) (21). Solutions for CD and UV-Tm measurements were prepared by diluting the stock nucleotide solutions (final concentration of 36 µmol/L) and the comb-type copolymer solution at various P/D ratios into buffer II. The mixtures were heated at 90 °C for 30 min, gradually cooled, and allowed to stand for 16 h at room temperature. UV spectra and UV melting curves were recorded with a Beckman DU-640 spectrometer equipped with a micromelting temperature apparatus (Beckman). The UV melting curves were recorded at 0.2 K/min as heating and cooling rates. The differential absorbance (∆A ) A260 - A340) was calculated to correct for baseline shift. The first derivative [d(∆A)/dT] was calculated from the melting curve data. Peak temperatures in the derivative curves were designated Tm. CD spectra were measured with a Jasco model J-600 spectropolarimeter. Each spectrum shown was the average of more than three scans that have been smoothed. Analysis of DNA-Polymer Complex Formation by Gel Electrophoresis. An increasing amount of each of the copolymers was added to 3 µg of pSV plasmid DNA (6.8 kbp) in buffer I from a stock solution. The mixture was diluted with buffer I to a final plasmid concentration of 43.4 µg/mL, followed by incubation for 24 h at room temperature. The mixture was then analyzed by running it on a 1% agarose gel in Tris-acetate buffer (40 mM, pH 8.0) containing 1 mM EDTA. The gel was then stained with ethidium bromide to visualize the DNA bands. The complex formation between a 20-mer oligonucleotide (ODN) and the graft copolymer was analyzed as follows. The ODN (500 ng) in 50 mM Tris-acetate (pH 7.0), 100 mM NaCl, and 10 mM MgCl2 was heated at 65 °C for 10 min to disrupt self-associated chains and then quickly cooled on ice. The copolymer was then added at the indicated P/D ratio and the mixture incubated for 12 h at room temperature. The mixture was then electrophoresed on a 15% polyacrylamide gel for 16 h at constant voltage (8 V/cm). The resulting bands were visualized by staining with Stain-all (Nacalai Tesque, Inc., Kyoto, Japan). A competition assay was also performed as described above, except that the ODN was incubated with or without copolymer at a P/D ratio of 1. Samples were electrophoresed in the presence or absence of an excess (ca. 10 times) amount of sonicated calf thymus DNA. RESULTS AND DISCUSSION

Successful Preparation of PLL-g-Dex with High Coupling Efficacy. Semitelechelic prepolymers which

Maruyama et al.

Figure 2. GPC profiles of PLL, Dex, and the resulting copolymers. Copolymers prepared from Dex having a Mn of 2600 (a) and a Mn of 5900 (b) were analyzed on Waters Ultrahydrogel 250 and 500 columns connected with a multiangle light scattering detector and a differential refractive index (RI) detector (see the text for details). An aqueous solution containing 0.5 M acetic acid and 0.2 M Na2SO4 was used as a mobile phase at a rate of 1.0 mL/min.

have a reactive group at their one chain end are required in the preparation of graft copolymers by the polymer coupling method. Polysaccharides are suitable prepolymers for graft copolymer preparation, since most polysaccharides possess one reductive end. Their semitelechelic character is preserved even after chemical or enzymic hydrolysis of the glycosyl bonds of the polysaccharide chains. Thus, by combining the hydrolysis and coupling reactions, we can obtain graft copolymers having polysaccharide chains with various molecular weights. In this study, the reductive end of Dex was directly coupled to the -amino groups of PLL by a reductive amination reaction using NaBH3CN as a reductant. Figure 2 shows GPC profiles of PLL, Dex, and the copolymers. These reactions show an apparent increase in molecular weight. Furthermore, since there is no corresponding Dex peak in the copolymer formulations, they can be assumed to be free of unreacted Dex. The results of the coupling reaction are summarized in Table 1 with the numberaverage molecular weight of the polymers estimated by static light scattering. At a lower feed ratio (70% regardless of the molecular weight of Dex and solvents used in the coupling process. The polymer composition calculated from the molecular weight was in good agreement with that determined from 1H NMR measurements (Figure 3). This result indicated that no detectable side reactions that may cause cross-linking or degradation of the resulting copolymers occurred. As reported elsewhere, the coupling reaction between PLL and other polysaccharides, including amylose (20), hyaluronic acid (22), and arabinogalactane (manuscript in preparation), were found to be successful, indicating the validity of the coupling polysaccharide conjugates with PLL. Turbidity of the DNA/Copolymer Mixture and the Melting Behavior of DNA in the Mixture. Polycationic homopolymers such as polylysine and polyarginine form precipitates or coacervates when they are mixed with polyanions such as DNA in aqueous media. The strong electrostatic attraction between oppositely charged macromolecules causes them to be drawn together and

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Bioconjugate Chem., Vol. 9, No. 2, 1998 295

Figure 3. 1H NMR spectra of PLL-g-Dex copolymers in D2O. The indicated run numbers were the same as those indicated in Table 1.

Figure 4. Turbidity of DNA/polymer mixtures (P/D ratio ) 1) in phosphate-buffered saline (PBS). To a 30 µg/mL sonicated calf thymus DNA solution in PBS was added a 1 mg/mL PBS solution of graft copolymers prepared with Dextran 4 (Mn ) 2600, O) or Dextran T-10 (Mn ) 5900, b) at a P/D ratio of 1. The mixture was diluted with buffer I to a final DNA concentration of 27.4 µg/mL. After the mixture was vigorously mixed with a vortex mixer, the turbidity of the solution was measured as the optical density (OD) at 340 nm.

react ionically, releasing their associated counterions as free salt (1, 23). The counterion release caused a significant loss in solubility due to a loss in osmotic pressure of the complex. The important characteristic of the comb-type graft copolymer is the fact that they form soluble complexes with DNAs as estimated by turbidity measurements of DNA/graft copolymer mixtures (Figure 4). The mixture of linear polylysine and DNA shows significant turbidity in 10 mM PBS containing 150 mM NaCl; however, the turbidity decreases with an increasing degree of grafting of Dex side chains. No turbidity is observed at higher degrees of grafting even after storage for 4 weeks at 4 °C. The grafted dextran chains seem to be responsible for protecting the DNApolycation complex from self-aggregation and precipitation. In support of our argument, we have seen, using fluorescence microscopy, that this protective effect also occurs in large (166 kbp) DNA sequences of T4 phage (data not shown). The solubility of such complexes minimized the effect of turbidity and/or precipitation and enabled us to evaluate their interaction with DNA by UV-Tm measurements. Figure 5 shows the UV-Tm curves of doublehelical poly(dA)‚poly(dT) in the absence or presence of the comb-type copolymers with various Dex contents. Poly(dA)‚poly(dT) alone undergoes double helix to single

Figure 5. UV-Tm profiles (upper panel) and its derivative curves (lower panel) of the poly(dA)‚poly(dT) duplex in the presence (P/D ) 1) of PLL-g-Dex copolymers having various degrees of grafting and lengths of Dex in buffer II. The differential absorbance (∆A ) A260 - A340) was plotted. Poly(dA) and poly(dT) were dissolved in buffer II. To a 1/1 mixture of poly(dA) and poly(dT) was added the copolymer at a P/D ratio of 1. After dilution to a final DNA concentration of 36 (bp) µmol/L with buffer II, the mixtures were heated at 90 °C for 30 min, cooled to room temperature, and allowed to stand for 16 h. The UV-Tm curves were recorded at 0.2 K/min as heating and cooling rates with a DU-640 spectrometer (Beckman) equipped with a micro-melting temperature apparatus. The differential absorbance (∆A ) A260 - A340) was used to correct for background variation. First-derivative curves were obtained from absorbance data.

strand transition at 72 °C in PBS. In the presence of the comb-type copolymers either with lower-molecular weight Dex, PLL-Dex(2600-7, -14, and -34) (Figure 5a), or with lower Dex content, PLL-Dex(5900-7) (Figure 5b), the complete melting of the double helix was not observed. In the presence of PLL-Dex(5900-7), although partial melting of the double-helical DNA to single strands was observed, reassociation of the ssDNA to form the double helix was incomplete in the cooling process. These results suggest that copolymers having lower degrees of grafting or shorter Dex chains interact firmly with the DNAs and prevent them from reversible duplexsingle strand transition. This is in agreement with the previously reported finding that polycations such as polylysine and polyarginine irreversibly interacted with DNA (3, 4). Like PLL homopolymers, the PLL backbone

296 Bioconjugate Chem., Vol. 9, No. 2, 1998

in the copolymers with a lower degree of grafting is assumed to interact irreversibly with DNA, leading to considerable stabilization of DNA double helices or inhibition of ssDNAs to form double helices. In other words, the Dex chains of these copolymers do not considerably hinder the interaction between DNA and the PLL backbone; however, they effectively increase the solubility of the complex. As seen in Figure 5, UV absorbance at 260 nm that is attributed to DNA in the mixture is decreased compared with that of free DNA. Similar behavior was previously observed in DNA/PLL mixtures (24), although significant turbidity was detected in this case. It seems that the polycationic backbone triggers a conformational change of the DNA to a dense globular structure (25-27), leading to a loss in UV absorption. However, we believe that further study on the assembly process between DNA and these copolymers is needed to understand the exact nature and behavior of the resulting complex. In contrast to the results obtained with the graft copolymers with lower degrees of grafting, the doublehelical DNA undergoes complete melting and reassociation when associated with the graft copolymer [PLLDex(5900-21)] with a higher degree of grafting (Figure 5b). The Tm of the dsDNA increased by more than 15 °C in the presence of an electrostatically equivalent amount ([amino group]polymer/[phosphate group]DNA ) 1) of PLLDex(5900-21). It can be concluded that PLL-Dex(590021) is capable of thermally stabilizing the poly(dA)‚poly(dT) double helices without interfering with their interpolynucleotide recognition and assembly. The abundant Dex graft chains (90 wt %) in PLL-Dex(5900-21) may prevent close association of the DNA to the PLL backbone, leading to weak PLL-DNA interactions. This weak interaction still allows the copolymer to thermally stabilize the DNA double helices possibly by reducing the electrostatic repulsion between negatively charged DNA strands. PLL-Dex(2600-34), for which the degree of grafting is higher than that of PLL-Dex(5900-21), does not allow the DNA to melt. Thus, both the degree of grafting and the length of graft chains are parameters by which we could regulate reversible melting of dsDNA. Stabilizing Effect of PLL-Dex(5900-21) on the Poly(dA)‚Poly(dT) Double Helix. The stabilizing effect of the graft copolymer, PLL-Dex(5900-21), having a higher Dex content was further evaluated by changing the polymer/DNA ratio. Figure 6 shows the copolymer/ DNA ratio ([amino group]polymer/[phosphate group]DNA ) P/D) effects on the melting of the poly(dA)‚poly(dT) double helix in the presence of PLL-Dex(5900-21). The melting temperature of the dsDNA gradually increases with an increasing P/D ratio. It is notable that an excess amount of the copolymer did not affect the reversibility of the DNA melting and reassociation. Since the ionic strength of the media is a factor that affects interpolyion interaction, we assessed the melting behavior of the double helix in a low-salt medium. Figure 7 shows the melting profiles of poly(dA)‚poly(dT) duplex in 1/100 diluted buffer I (0.1 mM PBS with 1.5 mM NaCl). Under these conditions, poly(dA)‚poly(dT) alone melted at 48 °C and did not exhibit obvious reassociation in the cooling process, indicating a considerably slower rate in doublehelix formation. Upon addition of the copolymer, the melting temperatures significantly increased and doublehelix reassociation also became more prominent. We concluded that the copolymer permits reassociation of DNA even under low-ionic strength conditions and promotes annealing of dsDNA. In the presence of a small amount of the copolymer, the DNA Tm surpassed 45 °C,

Maruyama et al.

Figure 6. P/D ratio dependency of thermal dissociation and reassociation of poly(dA)‚poly(dT) in the presence of PLL-Dex(5900-21) in buffer II: (a) change in absorbance at 260 nm and (b) first-derivative curves. The measurements were performed according to the same procedure described in Figure 5, except using different P/D ratios.

Figure 7. P/D ratio dependency of dissociation and reassociation of poly(dA)‚poly(dT) in the presence of PLL-Dex(5900-21) in 1/100 diluted PBS: (a) change in absorbance at 260 nm and (b) first-derivative curves The measurements were performed according to the same procedure as that described in Figure 6, except that the 1/100 diluted buffer II was used in these experiments.

leading to the same Tm observed in undiluted buffer I. The Tm value reached a plateau at a P/D ratio of 1, supporting the assumption that the graft copolymer and DNA form a stable association in buffer I. Further evidence supporting stable graft copolymer association is shown in Figures 6 and 7, where poly(dA)‚poly(dT) melted in a bimodal manner at a P/D ratio of 15, the CD signal was considerably different when compared to that of free DNA. The change in CD signals in the PLL/DNA mixtures has been explained by distortion and base tilting of the B conformation (3). X-ray diffraction results, however, indicated that DNA in these complexes remains essentially in the untilted B conformation, characteristic of free DNA in solution (3). The abnormal CD pattern was attributed to micellar or liquid crystal structure in the complex. CD signals observed at a wavelength of >300 nm indicated the formation of chiral aggregates (28-31). On the other hand, the graft copolymer does not induce a significant change in CD signals as shown in Figure 8b, suggesting only a slight effect on the DNA secondary structure. Similar results were also obtained even in the presence of large excesses of the copolymers. The CD measurements were also carried out in 1/100 diluted buffer I.

Although the PLL/DNA mixture was a transparent solution, probably due to a lower salting out effect, modified CD signals were recorded at all P/D ratios as shown in Figure 8c. The copolymer did not induce a significant change in CD signals even in low-ionic strength media (Figure 8d), in which ionic interactions should be significantly enhanced. The CD study was further carried out with the poly(dA)‚poly(dT) duplex as shown in Figure 9. Results similar to those obtained with calf thymus DNA were observed; namely, PLL-Dex(5900-21) addition did not significantly change the CD signals of poly(dA)‚poly(dT). It was confirmed that the graft copolymers with higher Dex content did not change the secondary structure of DNA double helices, while it elevated the Tm. Gel Electrophoresis of the DNA/Copolymer Mixture. The stability of the complex between DNA and the graft copolymer, PLL-Dex(5900-21), was assessed by gel electrophoresis of plasmid DNA (6.8 kbp) or a 20-mer oligodeoxynucleotide (ODN). As shown in Figure 10, migration of the plasmid DNA slowed as the P/D ratio increased. The graft copolymer completely inhibited migration of the plasmid DNA into the agarose gel at a nearly electrostatically equivalent point. Since the molecular weight of polyelectrolytes should be a factor which determines the stability of its IPECs, we then evaluated the complex of PLL-Dex(5900-21) with oligonucleotides. The migration of a 20-mer ODN (Figure 11A), which bears 19 phosphate anions in a molecule, into a 15% polyacrylamide gel was also impeded by the copolymer at a P/D ratio of 1 as shown in Figure 11B. PLL-Dex(5900-21) forms a stoichiometric complex even with a 20mer ODN which bears considerably fewer negative charges than the plasmid DNA. Although the interaction between PLL-Dex(5900-21) and DNA seemed to be weaker than the interaction between the PLL homopolymer and the copolymers with a lower degree of grafting or length, it retained an ability to form a relatively stable complex with DNA. The property of the complex was further estimated by a gel electrophoresis assay where an excess of thymus DNA (t-DNA) was added to the preformed complex. The ODN was completely and rapidly released from the complex by the addition of excess t-DNA (Figure 11C), while slight dissociation was observed with the PLL/ODN mixture (data not shown). Thus, PLL-Dex(5900-21)

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Figure 10. Analysis of complex formation between plasmid DNA (pSV) and PLL-Dex(5900-21) by agarose gel electrophoresis. The increasing amount of the copolymer was added to 3 µg of pSV plasmid (6.8 kbp) in PBS from a stock solution. The mixture was diluted with PBS to a final plasmid concentration of 43.4 µg/mL, followed by incubation for 24 h at room temperature. The mixture was then analyzed on a 0.8% agarose gel in Tris-acetate buffer (40 mM, pH 8.0) containing 1 mM EDTA. The plasmid was finally stained with ethidium bromide to visualize it. M marks the columns with low- and high-molecular weight markers.

stably retains the ODN but allows rapid exchange between DNA molecules. This unique behavior of the copolymer seems to be related to the reversible dissociation and association of the DNA duplex and triplex. The copolymer thus allows DNA molecules in the complex to recognize and hybridize to each other. CONCLUSION

Although the graft copolymer forms a soluble complex with DNA, physicochemical properties of DNA in the complex were significantly changed with the degree of grafting and length of graft chains. Graft copolymers with lower degrees of grafting firmly interacted with DNA, and probably condensed DNA into a globular conformation. This phenomenon was supported by a fluorescent dye exclusion assay using ethidium bromide and fluorescence microscopic observation of giant T4 DNA (data not shown). However, graft copolymers with higher degrees of grafting increased the Tm of the poly(dA)‚poly(dT) duplex by nearly 20 °C without affecting the reversible dissociation and reassociation of dsDNA. DNA associated with graft copolymers considerably accelerated hybridization of DNA strands in low-ionic strength media. The copolymers with higher degrees of grafting did not induce severe conformational changes of DNAs; furthermore, they still maintained the ability to form stoichiometric complexes with ODN. Thus, it can be concluded that grafting hydrophilic chains to PLL allows the regulation of the physicochemical properties of DNA-PLL copolymer complexes. Several conjugates between polycations and biological ligands have been examined as carriers of DNA, including genes and ODNs. DNA delivery by these carriers, however, lacks high efficiency. To improve DNA delivery using such conjugates, it is crucial to understand the structure-function relationships of DNA carriers and their complexes with DNA. Regulation of physicochemical properties and assembling structures of DNA-carrier complexes with graft copolymers may provide new knowledge for effective DNA delivery and may lead to remarkable progress in this field. We have recently found that the PLL graft copolymers with cell-specific polysaccharide chains permits cell-specific delivery and expression of a

Figure 11. Analysis of complex formation between oligodeoxynucleotide and PLL-Dex(5900-21) by polyacrylamide gel electrophoresis. (A) Nucleotide sequence of the 20-mer oligonucleotide (ODN). (B) Analysis of polymer-ODN complex formation. Polymer-DNA complex formation was analyzed according to the protocol shown above the gel and described in details in Materials and Methods. The ODN was heat-treated (HT) and incubated with (lanes 2-5) or without (lane 1) PLLDex(5900-21) at the indicated P/D ratio for 12 h at room temperature (RT). The mixtures were then separated by electrophoresis on a 15% polyacrylamide gel, and the gel was stained with Stain-all. An arrow indicates the position of the 20-mer ODN. (C) Competition assay. The ODN was incubated with (+) or without (-) PLL-Dex(5900-21) at a P/D ratio of 1. Just before the gel electophoretic analysis, thymus DNA (T-DNA) was added (lane 4). T-DNA alone was applied in lane 1. The position of the ODN is indicated by an arrow.

foreign gene in vivo (22). Furthermore, these copolymers may have several applications in which internucleotide recognition and/or supramolecular formation are involved. ACKNOWLEDGMENT

We are grateful to Dr. Horst von Recum for his valuable discussion. This work was supported in part by a grant-in-aid (09750967) for scientific research from the Ministry of Education, Science, Culture, and Sports of Japan. LITERATURE CITED (1) Michaels, A. S., and Miekka, R. G. (1961) Polycationpolyanion complexes: Preparation and properties of poly(vinylbenzyltrimethylammonium) poly-(styrenesulfonate). J. Phys. Chem. 65, 1765-1773.

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